A leading cause of pneumonia and related morbidity and mortality is the mucosal pathogen, Streptococcus pneumoniae. Beginning at the upper respiratory mucosa, infection begins with colonization which can then be complicated by pneumonia and invasive bloodstream infections. The most prominent antibody in these mucosal sites is the IgA1 subclass. IgA1 antibodies to the pneumococcal capsule, its primary virulence factor, are generated in response to colonization, symptomatic infection and vaccination. The variable region of IgA1 binds to the organism and the constant region binds to phagocytes (e.g., alveolar macrophages and neutrophils). A pneumococcal enzyme, IgA1 protease, is expressed on the surface of the bacteria. IgA1 bound to the capsule is cleaved by the protease at the bridging hinge between the variable and constant region, thereby inhibiting the ability of IgA1 to support phagocytosis, killing and clearance of the organism. Residual variable regions that remain on the surface modify the bacteria's surface and enhance binding to epithelial cell receptors, likely promoting colonization with the organism. This subversion of the protective host response to S. pneumoniae predisposes older veterans to increased risk for serious infection. Preventing the bacteria's inactivation of the host's response can be achieved by a subset of patients with invasive pneumococcal disease who generate IgG in serum that neutralizes the protease's ability to cleave IgA. Moreover, we have generated murine monoclonal antibodies (mMabs) that bind and some neutralize the protease. We propose to advance our understanding of the structure-function relationships of the protease and consider the feasibility of advancing this protein as a primary or adjunctive vaccine candidate. In this context, we Hypothesize that: 1) Neutralizing antibodies to IgA1 protease recognize conserved epitopes on the enzyme. 2) Protease-neutralizing antibodies with human IgA1 and prevent epithelial cell binding in vitro and colonization with intranasal challenge in vivo by inhibition of IgA1 cleavage. 3) Antibodies to IgA1 protease protect mice against fatal mucosal infection with S. pneumoniae indirectly by inhibiting cleavage of human IgA1 bound to the bacterial surface and directly by surface binding and mediating phagocytosis of the organism. To address these Hypotheses, we propose to pursue the following Specific Aims: Aim 1. Characterize the epitopes targeted by protease-neutralizing monoclonal antibodies (Mabs) and their genetic conservation. Aim 2. Characterize the epitopes targeted by protease-neutralizing monoclonal antibodies (Mabs) and their genetic conservation. Aim 3. Determine the ability of protease-specific Mab's to protect against fatal infection after mucosal challenge in vivo and the mechanisms underlying protection. This work is directed to determine the targets of the protease-neutralizing antibodies, the geographic and molecular diversity of pneumococcal proteases to consider the generalizability of these investigations, and the ability of protease binding and neutralization on IgA1 effector functions to prevent colonization and to support phagocytosis and killing of S. pneumoniae both in vitro and in vivo in mouse models. These studies provide important basic and pre-clinical data for considering the role of the protease in vaccine disease prevention and, potentially, therapy.